MicroRNAs play central roles in controlling gene expression in human cells. Sequencing data show that many miRNAs are produced at different levels and as multiple isoforms that can vary in length at their 5' or 3' ends, but the biogenesis and functional significance of these RNAs are largely unknown. We show here that the human trans-activation response (TAR) RNA binding protein (TRBP), a known molecular partner of the miRNA processing enzyme Dicer, changes the rates of pre-miRNA cleavage in an RNA-structure-specific manner. Furthermore, TRBP can trigger the generation of iso-miRNAs (isomiRs) that are longer than the canonical sequence by one nucleotide. We show that this change in miRNA processing site can alter guide strand selection, resulting in preferential silencing of a different mRNA target. These results implicate TRBP as a key regulator of miRNA processing and targeting in humans.

During RNA interference and related gene regulatory pathways, the endonuclease Dicer cleaves precursor RNA molecules to produce microRNAs (miRNAs) and short interfering RNAs (siRNAs). Human cells encode a single Dicer enzyme that can associate with two different double-stranded RNA (dsRNA)-binding proteins, protein activator of PKR (PACT) and trans-activation response RNA-binding protein (TRBP). However, the functional redundancy or differentiation of PACT and TRBP in miRNA and siRNA biogenesis is not well understood. Using a reconstituted system, we show here that PACT and TRBP have distinct effects on Dicer-mediated dsRNA processing. In particular, we found that PACT in complex with Dicer inhibits the processing of pre-siRNA substrates when compared with Dicer and a Dicer-TRBP complex. In addition, PACT and TRBP show non-redundant effects on the production of different-sized miRNAs (isomiRs), which in turn alter target-binding specificities. Experiments using chimeric versions of PACT and TRBP suggest that the two N-terminal RNA-binding domains of each protein confer the observed differences in dsRNA substrate recognition and processing behavior of Dicer-dsRNA-binding protein complexes. These results support the conclusion that in humans, Dicer-associated dsRNA-binding proteins are important regulatory factors that contribute both substrate and cleavage specificity during miRNA and siRNA production.

RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5' ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.

Bispecific antibodies have great potential to be the next-generation biotherapeutics due to their ability to simultaneously recognize two different targets. Compared to conventional monoclonal antibodies, knob-into-hole bispecific antibodies face unique challenges in production and characterization due to the increase in variant possibilities, such as homodimerization in covalent and noncovalent forms. In this study, a storage- and pH-sensitive hydrophobic interaction chromatography (HIC) profile change was observed for the hole-hole homodimer, and the multiple HIC peaks were explored and shown to be conformational isomers. We combined traditional analytical methods with hydrogen/deuterium exchange mass spectrometry (HDX MS), native mass spectrometry, and negative-staining electron microscopy to comprehensively characterize the hole-hole homodimer. HDX MS revealed conformational changes at the resolution of a few amino acids overlapping the C_H2-C_H3 domain interface. Conformational heterogeneity was also assessed by HDX MS isotopic distribution. The hole-hole homodimer was demonstrated to adopt a more homogeneous conformational distribution during storage. This conformational change is likely caused by a lack of C_H3 domain dimerization (due to the three "hole" point mutations), resulting in a unique storage- and pH-dependent conformational destabilization and refolding of the hole-hole homodimer Fc. Compared with the hole-hole homodimer under different storage conditions, the bispecific heterodimer, guided by the knob-into-hole assembly, proved to be a stable conformation with homogeneous distribution, confirming its high quality as a desired therapeutic. Functional studies by antigen binding and neonatal Fc receptor (FcRn) binding correlated very well with the structural characterization. Comprehensive interpretation of the results has provided a better understanding of both the homodimer variant and the bispecific molecule.

The immunopathological states of the brain induced by bacterial lipoproteins have been well characterized by using biochemical and histological assays. However, these studies have limitations in determining functional states of damaged brains involving aberrant synaptic activity and network, which makes it difficult to diagnose brain disorders during bacterial infection. To address this, we investigated the effect of Pam₃CSK₄ (PAM), a synthetic bacterial lipopeptide, on synaptic dysfunction of female mice brains and cultured neurons in parallel. Our functional brain imaging using PET with [¹⁸F]fluorodeoxyglucose and [¹⁸F] flumazenil revealed that the brain dysfunction induced by PAM is closely aligned to disruption of neurotransmitter-related neuronal activity and functional correlation in the region of the limbic system rather than to decrease of metabolic activity of neurons in the injection area. This finding was verified by in vivo tissue experiments that analyzed synaptic and dendritic alterations in the regions where PET imaging showed abnormal neuronal activity and network. Recording of synaptic activity also revealed that PAM reorganized synaptic distribution and decreased synaptic plasticity in hippocampus. Further study using in vitro neuron cultures demonstrated that PAM decreased the number of presynapses and the frequency of miniature EPSCs, which suggests PAM disrupts neuronal function by damaging presynapses exclusively. We also showed that PAM caused aggregation of synapses around dendrites, which may have caused no significant change in expression level of synaptic proteins, whereas synaptic number and function were impaired by PAM. Our findings could provide a useful guide for diagnosis and treatment of brain disorders specific to bacterial infection.SIGNIFICANCE STATEMENT It is challenging to diagnose brain disorders caused by bacterial infection because neural damage induced by bacterial products involves nonspecific neurological symptoms, which is rarely detected by laboratory tests with low spatiotemporal resolution. To better understand brain pathology, it is essential to detect functional abnormalities of brain over time. To this end, we investigated characteristic patterns of altered neuronal integrity and functional correlation between various regions in mice brains injected with bacterial lipopeptides using PET with a goal to apply new findings to diagnosis of brain disorder specific to bacterial infection. In addition, we analyzed altered synaptic density and function using both in vivo and in vitro experimental models to understand how bacterial lipopeptides impair brain function and network.

Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity "broad-spectrum" therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspects of relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a wide range of high-priority targets (74 in total) that could be modified to improve patient outcomes. For these targets, corresponding low-toxicity therapeutic approaches were then suggested, many of which were phytochemicals. Proposed actions on each target and all of the approaches were further reviewed for known effects on other hallmark areas and the tumor microenvironment. Potential contrary or procarcinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixed evidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of the relationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. This novel approach has potential to be relatively inexpensive, it should help us address stages and types of cancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for future research is offered.